Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• • C—CHEMISTRY; METALLURGY
  • C05—FERTILISERS; MANUFACTURE THEREOF
  • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
  • C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
  • C05F17/80—Separation, elimination or disposal of harmful substances during the treatment
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/005—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor after treatment of microbial biomass not covered by C12N1/02 - C12N1/08
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/08—Reducing the nucleic acid content
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/141—Feedstock
  • Y02P20/145—Feedstock the feedstock being materials of biological origin
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
  • Y02W30/00—Technologies for solid waste management
  • Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse

Definitions

• the present invention relates to a process for fragmentation of fungal, bacterial or yeast cell DNA and for inactivating any residual antibiotics directly in fermentation biomass.
• biomass is produced which has to be disposed of.
• the said biomass is rich in nitrogen, phosphorus, potassium, carbon and organic compounds, rendering it ideal for transformation into fertilizer, such mass being bioavailable for ready assimilation by soil microflora and having no undesirable ground-contaminating substances.
• the high percentage of organic-biological components makes the biomass the ideal fertilizer for soil intended for those organic crops increasingly required by the fruit and vegetable market.
• the residual biomass from the said fermentations contains potentially transgenic genetic material which, if deriving from antibiotic production fermentation, also contains residual antibiotics the presence of which in a fertilizer could damage, modify or alter the soil microflora.
• the potentially transgenic material is formed from the double helix of the fungal, bacterial or yeast DNA which could give rise to the formation of genetically modified organisms (GMO).
• GMO genetically modified organisms
• Residual antibiotic in fermentation biomass can be easily monitored, until its disappearance, by well known high pressure liquid chromatography (HPLC), which requires no detailed explanation.
• HPLC high pressure liquid chromatography
• the main object of the present invention is therefore to provide a process of simple and reliable implementation on an industrial scale which both enables the double helix of fungal, bacterial or yeast DNA present in fermentation biomass to be fragmented to below 1000 base pairs, and enables any antibiotic residue in said biomass to be eliminated.
• the process consists of a treatment which destroys both the DNA and any residual antibiotic
• operative parameters such as the agitation rate during heating and acid addition modalities are of little importance in attaining the stated object. It has however been noted that heating must be continued for a longer time if the temperature is low (close to +50°C) than if the temperature is high (close to +110°C), to enable the heat administered at acid pH to act on the entire mass, so contributing to hydrolyze amides and a large part of the DNA and antibiotic esters: in particular, in penicillins and cephalosporins the thermolability of the amide bond at acid pH favours inactivation of the residual antibiotic active principle, with opening of the betalactam ring, on the basis of the heating time, which is variable according to the stability of the antibiotic subjected to inactivation treatment.
• the required heating time duration can be easily verified by analyzing the treated biomass by the aforesaid methods, with regard both to the dimensions of the extracted and analyzed DNA fragments and the presence of residual antibiotic.
• the temperature used in the process of the invention is between +80° and +100°C;
• the preferred acids used in aqueous solution are those of the group comprising acetic acid, methanesulphonic acid, phosphoric acid and sulphuric acid (which are compatible with the presence of acetates, phosphates and sulphates in agricultural soil); while neutralization is effected with bases chosen from the group consisting of calcium, sodium, potassium, ammonium hydroxide, or triethylamine, diethylamine and similar organic bases.
• a fungal biomass resulting from a standard industrial fermentation of Cephalosporin C was acidified to pH 1.0 with 96% H 2 SO 4 .
• the acidified mass was maintained heated to 90°C for 90 min, then cooled to ambient temperature and neutralized to pH 7 with Ca(OH) 2 .
• Example 2 Proceeding as in Example 1 but acidifying to pH 1.0 with H 3 PO 4 and heating to 90°C for 60 min, the test for DNA showed the absence of fragments greater than 500 base pairs.
• Example 2 Proceeding as in Example 1 but acidifying firstly to pH 2.0 with a concentrated solution of H 3 PO 4 then adjusting the pH to 1.0 with an aqueous 30% H 2 SO 4 solution, and then heating to 90°C for 60 min, no DNA fragments greater than 500 base pairs were observed.
• Example 3 Operating as in Example 3 on a sample of fungal biomass from Cephalosporium acremonium grown in a flask, after removing 50% supernatant liquid from the biomass (which is hence more than 50% concentrated), results similar to those indicated in Example 3 were obtained.
• Results for DNA and residual antibiotic similar to the aforestated are obtained if the sample is heated to 110°C for 60 min.
• a sample of spent mycelium (5 litres of fungal biomass) withdrawn on termination of fermentation from an industrial Cephalosporin C process, with residual Cephalosporin C 1.4 g/I, was transferred to a 10 litre boiler of AISI 316 steel fitted with a stirrer, heating jacket, manhole. thermometer and pH meter. 1 litre of water was added to the sample to increase its fluidity and improve stirring.
• Example 7 A sample of spent mycelium as in Example 7 was acidified to pH 1.01 with 230 ml of 85% phosphoric acid. The sample was then heated to 90°C for 300 min, cooled to ambient temperature, topped up to its initial volume, neutralized to pH 7.1 with diethylamine and analyzed for DNA and residual Cephalosporin C. Values comparable to those of Example 7 were found.
• Example 8 A sample of spent mycelium as in Example 8 was acidified to pH 2.02 with 56 ml of 85% H 3 PO 4 and then adjusted to pH 1.02 with 50 ml of 96% H 2 SO 4 . The sample was heated to 95°C for 180 min, cooled to ambient temperature, topped up to its initial volume and neutralized to pH 7.04 with Ca(OH) 2 . Analyses for DNA and residual antibiotic gave results comparable to those of Example 7.
• the diluted sample was transferred to a 10 litre AISI 316 steel boiler equipped as in Example 7.
• the resultant suspension was heated to 95°C for 360 min, cooled to ambient temperature, topped up to its initial volume by adding water (about 350 ml) and neutralized to pH 7.09 with 25% NH 4 OH.
• DNA and residual antibiotic analyses confirm the absence of base pairs > 500, with antibiotic ⁇ 1 ppm.
• a sample such as that described in this example was adjusted to pH 2.2 with 85% H 3 PO 4 and heated to 100°C for 450 min.
• the sample, cooled and neutralized with Ca(OH) 2 was analyzed for DNA and residual antibiotic, obtaining values comparable to the aforestated.
• the aforegiven examples describe the treatment for inactivating DNA and residual antibiotic in fungal and bacterial biomass from Cephalosporin C and Tylosin production.
• the same treatment can be implemented on fermentation masses using yeast or other fungal or bacterial strains used to ferment other antibiotic principles (such as Penem, Penam, Cepham, Cephem, Tetracyclines, Macrolides, Aminoglycosides).

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Cited By (7)

Citations (2)

• 2003
  • 2003-11-05 IT ITMI20032129 patent/ITMI20032129A1/it unknown
• 2004
  • 2004-10-27 EP EP20040025494 patent/EP1529766B1/fr not_active Not-in-force
  • 2004-10-27 ES ES04025494T patent/ES2255026T3/es active Active
  • 2004-10-27 AT AT04025494T patent/ATE316949T1/de active
  • 2004-10-27 DE DE200460000363 patent/DE602004000363D1/de active Active

Patent Citations (2)

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